Summary Abstract Predicting the efficacy of an antibiotic in a patient is a critical component of successfully treating infection. Currently, there is an over-reliance on susceptibility assays involving pure bacterial cultures and controlled growth conditions. These conditions are drastically different to those experienced by the bacteria during infection. Hence, these assays fail to account for extrinsic factors that influence antibiotic susceptibility in the infection environment. This contributes to unacceptable rates of treatment failure. Staphylococcus aureus is responsible for numerous difficult-to-treat infections and antibiotic treatment failure is common. The identification of novel extrinsic factors that significantly influence S. aureus antibiotic susceptibility in vivo will greatly improve our ability to predict antibiotic efficacy in patients, leading to improved treatment outcomes. These factors may include host interactions, nutrient and oxygen availability and interactions with other microorganisms during polymicrobial infection. Our preliminary studies demonstrate that P. aeruginosa produced molecules dramatically alter S. aureus antibiotic susceptibility. We find the production of these molecules is highly variable among P. aeruginosa clinical isolates. Preliminary experiments in mice suggest these interactions play an important role in determining antibiotic efficacy in vivo. We hypothesize that P. aeruginosa strongly influences S. aureus antibiotic susceptibility in patients during polymicrobial infection. In Aim 1, we propose to elucidate the molecular mechanisms responsible for this altered antibiotic efficacy. Elucidation of these mechanisms will improve our understanding of S. aureus antibiotic tolerance and may also lead to the development of novel adjuvants for the eradication of S. aureus populations. In Aim 2, to determine the importance of P. aeruginosa/S. aureus interactions in patients, we will examine clinical isolate pairs from burn and cystic fibrosis patients. In Aim 3, to determine the relevance of these interactions during infection, we will examine antibiotic efficacy against S. aureus mono-infection and during co-infection with P. aeruginosa using two complimentary mouse models of infection. Together, these experiments promise to reveal the role of bacterial interaction in determining the outcome of antibiotic treatment of S. aureus. This will facilitate the future development of more sophisticated, more accurate susceptibility determination and improved treatment outcomes in patients.